DESCRIPTION: The major goal of the proposed research continues to be elucidation of the mechanisms controlling the switch from latency to viral replication in Epstein-Barr virus (EBV) infected B lymphocytes. More specifically, this proposal focuses on the regulation of two linked EBV genes, BZLF1 and BRLF1, that are integrally involved in this switch. During the previous funding period, significant progress has been made, identifying the critical cis-elements involved in regulation the BZLF1 gene promoter (Zp), and the cellular factors that bind to these sites. In addition, two calcium response pathways have been identified that can trigger viral reactivation. However, only a partial picture exists for how transcription of the BZLF1 gene carefully analyzed. Thus, to identify and further define the cis-elements, cellular transcription factors and signaling pathways involved in regulating the BRLF1/BZLF1 gene locus we propose the following aims: Aim 1- Functional analysis of cis-elements involved in regulating Zp and Rp; 1a. investigate the interdependence of the BRLFA and BZLF1 genes; 1b. identify and characterize cis-elements involved in regulating induction of Rp; 1c. characterize role of Sp1, Sp2 and MEF2D binding to the Z1 domains in Zp; 1d. characterize and clone the cellular factor(s) binding to the negative locus; 2a. characterize the role of Zp and Zta in the induction of the viral lytic cycle; 2b. assess the impact of selected mutations in critical cis-elements involved in regulating Zp and R; Aim 3- Analysis of calcium responsive pathways involved in reactivation of EBV; 3a. determine the mechanism of NFAT induction of Zp through MEF2D; 3b. determine the target(s) of CaMKIV in the induction of Zp.